CN111330025B - Bionic microbubble ultrasound contrast agent and preparation method thereof - Google Patents
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- A—HUMAN NECESSITIES
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Abstract
The invention belongs to the field of medicine, and relates to a bionic lipid microbubble ultrasound contrast agent constructed by cell membranes and a preparation method thereof. The preparation method comprises the steps of firstly separating cells; extracting cell membranes; ultrasonically vibrating the cell membrane and phospholipid to prepare liposome mixed with the cell membrane and the phospholipid; and finally, introducing gas into the liposome, and mechanically oscillating to prepare the targeted microbubble ultrasound contrast agent. The bionic lipid microbubble ultrasound contrast agent disclosed by the invention integrates the biological characteristics of cell membranes into the ultrasound contrast agent, and breaks through the application range of the traditional microbubble ultrasound contrast agent. The preparation method of the bionic liposome has the advantages of natural targeting and easy clinical transformation.
Description
Technical Field
The invention relates to the field of ultrasonic molecular imaging and biomedical engineering, in particular to a bionic microbubble ultrasonic contrast agent and a preparation method thereof.
Technical Field
The ultrasonic molecular imaging is a molecular imaging method which takes a targeted microbubble contrast agent as a molecular probe and takes ultrasonic imaging as an imaging mode to carry out qualitative and quantitative research on the cellular and molecular level of the biochemical process of a living body, can improve the accuracy and sensitivity of ultrasonic diagnosis, and has important application prospect in the fields of diseases such as cardiovascular diseases, inflammatory diseases, tumors and the like.
The ultrasonic imaging has the advantages of simple and quick operation, real-time observation of the dynamic development of the vascular injury focus, economy and reduced economic pressure of patients. But the defects are that the ultrasonic imaging resolution ratio is low, and a contrast agent is needed to enhance the contrast ratio of the ultrasonic imaging and improve the resolution ratio.
Conventional ultrasound contrast agents are microbubbles encapsulated with lipids, albumin, and polymers. The ultrasound contrast agent using phospholipid as material, such as Sonovue (italy, Bracco) has been approved by FDA and widely used in clinical ultrasound diagnosis, has the characteristics of high safety, good stability, and capability of generating abundant harmonic signals, effectively improves the sensitivity of disease diagnosis, and has been written into a plurality of disease diagnosis and treatment application guidelines at present.
However, these conventional ultrasound microbubble contrast agents use exogenous substances as membrane materials, which not only increases the problem of biological safety, but also is difficult to achieve targeted imaging because the conventional ultrasound microbubble contrast agents lack natural targeting property and cannot specifically recognize adhesion molecules highly expressed in target tissues. In addition, the conventional ultrasound microbubble contrast agents have short circulation time in vivo due to the instability problem, and the application and development of the conventional ultrasound microbubble contrast agents in the aspect of molecular imaging are limited.
Disclosure of Invention
One of the purposes of the invention is to provide a bionic microbubble ultrasound contrast agent aiming at the defects that the existing microbubble ultrasound contrast agent lacks natural targeting and has poor stability.
The invention also aims to provide a preparation method of the bionic microbubble ultrasound contrast agent.
The bionic particle taking the cell membrane as the material has the characteristics of low immunogenicity, good biocompatibility, difficult rapid removal by a reticuloendothelial system, long cycle period, good stability and the like, and can increase the aggregation of specific adhesion molecules in a target tissue by activating the cell membrane to over-express the specific adhesion molecules. At present, the cell membrane has been used as a drug-coated membrane, but the research on preparing the ultrasound contrast agent by using the cell membrane is very limited.
The invention realizes the purpose through the following technical scheme:
in one aspect, the invention provides a microbubble ultrasound contrast agent, comprising a cell membrane and a liposome; the cell membrane is selected from mammalian blood cell membranes and the liposome is selected from phospholipids.
In some embodiments, the cell membrane is selected from the group consisting of rat, mouse, rabbit, dog, pig, or human blood cell membranes.
In some embodiments, the cell membrane is selected from one or more of a red blood cell, white blood cell, or platelet membrane.
In some embodiments, the cell membrane is selected from the group consisting of cell membranes of leukocytes.
In some embodiments, the phospholipid comprises one or more of Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA).
In some embodiments, the phospholipid comprises a combination of Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA).
In some embodiments, the phospholipid comprises Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA) in a molar mass ratio of 18: 1:1 to 5, more preferably 18: 1: 1.
in some embodiments, the mass ratio of liposomal phospholipid to cell membrane protein is (20-1000): 1.
in some embodiments, the mass ratio of liposome phospholipids to cell membrane proteins is (20-800): 1.
in some embodiments, the mass ratio of liposome phospholipids to cell membrane proteins is (40-700): 1.
in some embodiments, the mass ratio of liposomal phospholipid to cell membrane protein is 50: 1.
in some embodiments, the microbubble ultrasound contrast agent is 0.2 to 4.4 μm in size.
On the other hand, the invention also provides a preparation method of the lipid microbubble ultrasound contrast agent, which comprises the following steps: the cell membrane is mixed with the liposome and gas is introduced, and the mechanical oscillation forms lipid microbubbles containing the cell membrane.
In some embodiments, the method of making comprises the steps of:
s1, separating blood cells from peripheral blood;
s2, extracting cell membranes by using a differential centrifugation method;
s3, preparing liposome by a thin film-hydration method;
and S4, mixing the cell membrane prepared in the S2 with the liposome prepared in the S3, introducing gas, and mechanically oscillating to form the lipid microbubble containing the cell membrane.
In some embodiments, the preparation method, step S1, comprises the steps of:
s11, taking a blood sample in an anticoagulation blood collection tube;
s12, adding the blood cell separation liquid with the same volume as the blood sample in the S11 into a centrifuge tube, sucking the blood sample, paving the blood sample on the liquid level of the cell separation liquid, and centrifuging the blood sample for 20 to 40min at the temperature of between 18 and 22 ℃ at the temperature of 400-1000 g;
s13, after centrifugation in the step S12, sucking the needed blood cells in the separation liquid; preferably, the desired blood cells are leukocytes;
s14, adding lysis solution with the volume 2-5 times that of the blood cells sucked in the step S13, blowing, uniformly mixing, lysing for 5-20min, centrifuging for 5-15min at 200g-400g, and removing supernatant;
s15, repeating the step S14 once to obtain the needed blood cells;
s16, adding 5-15ml of cell washing liquid into the blood cells obtained in the step S15, uniformly mixing the cells, centrifuging for 5-15min at 200-400g, and removing the supernatant;
s17, repeating the step S16 for three times to obtain the purified blood cells.
In some embodiments, step S2 includes the steps of:
s21, adding 10-20ml of hypotonic lysis solution into the blood cells separated in the step S1 for resuspension, transferring the blood cells into a glass homogenizer, and homogenizing the blood cells on ice for 15-30 times to obtain homogenate.
In some embodiments, the hypotonic lysis solution comprises 30mM Tris-HCl pH7.5, 225mM D-mannitol, 75mM sucrose, 0.2mM EGTA, a protease inhibitor and a phospholipase inhibitor.
S22, adding the homogenate obtained in the step S21 into a centrifuge tube, centrifuging for 15-30min at 10,000g, sucking supernatant, centrifuging for 30-50min at 100,000g, discarding the supernatant, and leaving a precipitate.
S23, resuspending the precipitate obtained in the step S22 into a solution by using triple distilled water, freezing the resuspended solution at-70 to-90 ℃, and performing vacuum freeze-drying to obtain a blood cell membrane, and storing at-15 to-25 ℃.
In some embodiments, DiI dye is added while the pellet from step S22 is resuspended as a solution with triple distilled water.
In some embodiments, step S3 includes the steps of:
s31, uniformly mixing phospholipid and chloroform;
s32, forming a phospholipid film by the solution prepared in the step S31 under the vacuum action of a rotary evaporator;
s33, adding a hydration liquid into the phospholipid film prepared in the S32, and hydrating the phospholipid film in a hot water bath of a rotary evaporator to form a liposome; the hydration liquid is selected from double distilled water, sodium chloride or Phosphate Buffer Solution (PBS); preferably, the ratio of the hydration solution to the phospholipid is 3-8ml to 20 mg.
In some embodiments, in step S31, the chloroform to phospholipid ratio is 3-8ml to 20 mg.
In some embodiments, the dye DiO is added to the liposomes made from S31.
In some embodiments, the rotational speed of the rotary evaporator is 100-120rpm, and the temperature is 50-70 ℃, preferably 60 ℃ in step S32;
in some embodiments, in step S33, the hydration temperature is 50-60 ℃ and the hydration time is 20-40min, preferably 30 min;
in some embodiments, step S4 includes the steps of:
s41, gas replacement: the liposomes prepared in step S3 are mixed with the blood cell membranes prepared in step S2, and air is replaced by introducing gas.
S42, mechanically shaking the mixed solution obtained in the step S41 for 40-50S to form cell membrane bionic lipid microbubbles; preferably, the mixed solution is mechanically shaken for 45 s; preferably, the mechanical oscillation frequency is 3000-5000cpm, more preferably 4350 cpm;
in some embodiments, in step S41, the gas introduced is perfluoropropane C 3 F 8 (ii) a Preferably, the gas is passed in an amount of 5-15ml, more preferably 10 ml.
In another aspect, the invention also provides the application of the microbubble ultrasound contrast agent in the preparation of targeted ultrasound contrast agents.
In some embodiments, the microbubble ultrasound contrast agent targets a site of inflammation.
The method has the beneficial effects that:
the application range of the traditional microbubble ultrasound contrast agent is limited to non-targeted diagnosis imaging in blood circulation, and the lipid microbubble ultrasound contrast agent takes cell membranes as membrane materials, can integrate the biological characteristics of the cell membranes into the ultrasound contrast agent, has natural targeting property, and can identify inflammatory parts in a targeted manner. In addition, the preparation method overcomes the defects of low biocompatibility of the contrast agent prepared from a pure phospholipid material, low yield and poor stability of the contrast agent prepared from a pure cell membrane bionic material.
Drawings
FIG. 1 shows the structure of a leukocyte-mimicking lipid microbubble inverted fluorescence microscope;
in the figure, A represents a DiO-labeled lipid microbubble membrane, B represents a DiI-labeled cell membrane, C is a superimposed graph of A and B, and D is an image under a bright field of a simulated white cell membrane microbubble.
FIG. 2 shows the distribution of the particle size distribution (2A) and the Zeta potential (2C) of the control microbubbles, and the distribution of the particle size distribution (2B) and the Zeta potential (2D) of the leucocyte membrane-simulated lipid microbubbles, respectively.
Fig. 3 shows the flow results of the leukocyte membrane-simulated lipid microvesicles, wherein A, C is a control microvesicle distribution map and a bionic microvesicle distribution map, and B, D is a control microvesicle and a bionic microvesicle CD45 expression map.
FIG. 4 is the immunoblot results of lipid microvesicles mimicking leukocytes, indicating that the expression of biomimetic microvesicles, leukocyte membranes and total proteins of leukocytes are substantially identical. Mainly expresses CD47 and an inflammation-related ligand Mac-1/LFA-1, L-selectin and the like.
FIG. 5 is a targeted ultrasound image of the leucocyte membrane-mimicking lipid microbubble in an ischemia-reperfusion rat model and a control animal, in which there is no significant difference in ultrasound signal intensity when the control microbubble and the leucocyte membrane-mimicking lipid microbubble are injected; in an ischemia reperfusion animal model, the ultrasound signal intensity after injecting the leukocyte-mimicking microvesicles is higher than that of the control microvesicles.
FIG. 6 is an imaging diagram of bionic microbubble ultrasound contrast agents prepared by mixing leucocyte membrane proteins with liposomes in different mass ratios at different time points.
Detailed Description
The technical solutions of the present invention are further illustrated by the following specific examples, which do not represent limitations to the scope of the present invention. Insubstantial modifications and adaptations of the present invention by others based on the teachings of the present invention are within the scope of the invention.
Example 1: preparation of leucocyte-imitating microbubble ultrasound contrast agent
The rats were injected with LPS 1.5mg/kg intraperitoneally, and after 6 hours of sevoflurane inhalation anesthesia, the abdominal cavity was opened to expose the abdominal aorta for blood sampling in an anticoagulated blood collection tube. Another 50ml centrifuge tube is taken and added with the separation liquid of peripheral blood white blood cells (Solarbio) of rats with the same volume as the blood sample; blood samples were drawn using a Pasteur pipette, carefully spread on top of the surface of the separation medium and centrifuged at 1000g for 30 min. After centrifugation, two annular milky white cell layers appear in the centrifuge tube, the upper layer of cells is a mononuclear cell layer, and the lower layer of cells is a leucocyte layer. Carefully sucking the leucocyte layer in the separation solution by using a suction tube, adding erythrocyte lysate (Solarbio) with 3 times of cell volume, gently blowing and uniformly mixing, performing lysis for 10min, centrifuging for 10min at 300g, and discarding red supernatant; repeating the cracking step once to obtain the leucocyte. Adding 10ml of cell washing liquid into the obtained cells, uniformly mixing the cells, centrifuging for 10min at 250g, and removing supernatant; the washing step is repeated to obtain purified leukocytes.
The leukocyte membrane was extracted by differential centrifugation, and 12ml of hypotonic lysis solution (30mM pH7.5 Tris-HCl, 225mM D-mannitol, 75mM sucrose, 0.2mM EGTA, protease inhibitor and phospholipase inhibitor) was added to the above-obtained leukocytes to resuspend them, and transferred to a glass homogenizer, and homogenized on ice for 20 times. The resulting solution was added to a centrifuge tube and centrifuged at 10,000g for 20 min. The supernatant was aspirated and centrifuged at 100,000g for 40 min. The supernatant was discarded and the pellet resuspended in triple distilled water (appropriate amount of DiI dye was added if necessary). Freezing the solution at-80 deg.C, vacuum lyophilizing to obtain leukocyte membrane, and storing at-20 deg.C;
the liposome is prepared by a film hydration method, and 18mg of DPPC, 3.5mg of DSPE-PEG2000 and 1mg of DPPA (molar mass ratio is 18: 1: 1) are weighed and dissolved in 4ml of chloroform. Placing on a rotary evaporator to carry out vacuum drying to form a phospholipid film (60 ℃, 120 rpm); 4ml of PBS hydration solution was added to the phospholipid membrane and hydrated in a heated water bath on a rotary evaporator for 30min (60 ℃, 120rpm) to form liposomes.
Mixing the 400. mu.l liposome with 100. mu.l white cell membrane, and introducing C 3 F 8 Displacing air; the mixed solution was mechanically shaken (45s, 4350cpm) to form leukocyte membrane-mimicking lipid microbubbles. As a control, 500. mu.l of the above liposomes was aspirated and C was added 3 F 8 The control microbubbles were formed by mechanical shaking (45s, 4350cpm) after air displacement.
When preparing a cell membrane, adding a proper amount of fluorescent dye DiI, and adding dye DiO into liposome to form a bionic cell membrane lipid microbubble, and observing the microbubble structure under an inverted fluorescence microscope.
The results show that: the liposome has green fluorescence, the cell membrane has red fluorescence, and the two kinds of fluorescence are almost completely overlapped, which shows that the cell membrane is integrated on the lipid microbubble membrane, and the cell membrane-simulated lipid microbubble is successfully prepared.
Using dynamic light scatteringSystematically measuring the particle size and potential of the microbubbles, and diluting the microbubbles to 1X 10 7 3ml of the sample is added into a sample pool for measurement.
The results show that: the particle sizes of the control microvesicle and the leucocyte membrane-imitated lipid microvesicle are respectively 1.06 +/-0.02 mu m and 1.01 +/-0.02 mu m; the electric potentials are respectively-14.87 +/-1.57 mv and-27.07 +/-3.45 mv.
Example 2: detection of biomimetic microbubble-targeted ligands
The control and the biomimetic microvesicles prepared above were purified by centrifugation (2000rpm, 2min), the lower layer liquid was discarded, 500. mu.l PBS was added for resuspension, 4.5. mu.g CD45 flow antibody was added, and incubation was performed for 30min at room temperature. Then centrifuged again (2000rpm, 2min), the lower layer liquid was discarded to remove free antibody, 500. mu.l PBS was added for resuspension, and the solution was diluted to 10 5 And (4) detecting the expression of the microvesicle CD45 by flow cytometry.
The results show that: bionic Microvesicles (MB) m ) The expression level of CD45 (30.6%) was significantly higher than that of control Microvesicles (MB) con ) (3.34%) (FIG. 3), indicating that the biomimetic microvesicles carry the leukocyte membrane-specific protein CD 45.
The control and biomimetic microvesicles prepared above were purified by centrifugation (2000rpm, 2min), the lower layer liquid was discarded, and 500. mu.l PBS was added for resuspension. The microvesicles, the leukocytes extracted above and the leukocyte membrane species were lysed by adding 500. mu.l of RIPA lysate for 30min, followed by centrifugation (2000rpm, 2min) to obtain the supernatant. The expression condition of the specific protein is detected by adopting an immunoblotting method (Western blotting).
The results show that: leukocyte membrane-mimicking lipid Microvesicles (MB) m ) Leukocyte membrane (LEU) m ) Is basically consistent with the overall protein expression of Leucocytes (LEU) (FIG. 4), and mainly expresses a ligand CD47 avoiding recognition of reticuloendothelial cells, an inflammation-related ligand Mac-1/LFA-1, L-selectin and the like.
The results show that the bionic microvesicles prepared by the invention retain related proteins and ligands and have targeting property.
Example 3: ultrasonic imaging capability detection of bionic microbubble ultrasonic contrast agent
Establishing a rat liver ischemia-reperfusion injury (IRI) model: the rats are fasted for 8-12h before operation, and are anesthetized by intraperitoneal injection by using ketamine (60mg/kg) and xylazine (100mg/kg) compound liquid. Fixing rat on operating table, making abdominal incision, exposing abdominal cavity, dissociating hepatic portal blood vessel, clamping hepatic left lobe and middle lobe blood flow with vascular clamp, removing vascular clamp after 60min, and suturing incision. Suture operation after opening abdomen was performed for 0min in ischemia of control group.
After anaesthetizing, the animals were fixed on the operating table. The suture line is cut along the median abdominal incision, and the ultrasonic probe (color doppler ultrasonic diagnostic system, philips EPQ7, 5-12L high frequency linear array probe 12MHz) is fixed above the left and middle lobes of the liver, with the following imaging parameters: frequency 10MHz, gain 20-40dB, imaging depth 2.5-4.0cm, and mechanical index 0.07. The leukocyte-mimicking membrane microbubbles prepared in example 1 and the control microbubbles were diluted to about 10 8 100 μ l microbubbles were injected into the tail vein in random order, with 30min intervals between injections. Targeted ultrasound imaging was performed by a burst-reperfusion method, i.e. free and targeted bound microbubbles in the circulation were burst by a "flash" high mechanical index ultrasound (0.24, 1s) given 60s after microbubble injection, followed by 10s ultrasound image acquisition of free microbubbles in the circulation.
The results show that: the ultrasound imaging results were analyzed by Normalized Intensity Difference (NID), i.e., signal Intensity (pre-blast-post-blast)/background signal x 100%. In the control group of rats, tail vein injection of the control microvesicle and the leucocyte membrane-imitating microvesicle showed no significant difference in signal intensity (6.23% + -2.14% vs 8.23% + -1.12%). The signal intensity after injection of the targeting microvesicles was higher in IRI rats than the control microvesicles (8.20% ± 2.00% vs 18.19% ± 3.12%), demonstrating the ability of the leucocyte membrane-mimicking microvesicles to target sites of ischemia-reperfusion inflammation (fig. 5).
Example 4: effect of different method parameters on biomimetic microbubble ultrasound contrast Agents
At present, how to prepare bionic microbubbles with stable performance and good ultrasonic imaging effect is the most main obstacle in the field, and the stability and the ultrasonic imaging capability of phospholipids and cell membranes with different mass ratios are explored below.
Mixing leukocyte cell membrane and liposome phospholipid at different mass ratios (leukocyte cell membrane protein: phospholipid: 1: 0, 1:2, 1:5, 1:10, 1:50, 1:100, 1:300, 1:600), mixing well by ultrasonic vibration, introducing 10ml C 3 F 8 The gas displaces air and mechanically oscillates for 45s to form microbubbles. After 1000-fold dilution, in vitro stability assay was performed, 1ml of microbubbles was added to the mimetics and imaged at different time points (0, 10, 20, 30, 40, 50, 60min), and the stability and imaging ability of the microbubbles was observed.
The results show that: as shown in fig. 6, microbubble stability decreased with time; at the same time point, the microbubble stability tended to decrease as the proportion of white cell membranes increased. The stability and imaging effect of pure cell membranes are not good enough, and the requirements of ultrasonic imaging cannot be met. Leukocyte membrane protein: the mass ratio of liposome phospholipid is 1: when the content is 50-600, the stability of the micro-bubble is better.
Claims (28)
1. A microbubble ultrasound contrast agent, which is characterized by comprising a cell membrane and a liposome; the liposome takes phospholipid as a raw material and is prepared by a thin film-hydration method, the cell membrane is selected from white cell membranes, and the preparation method of the microbubble ultrasound contrast agent comprises the following steps: mixing leukocyte membrane and liposome, introducing gas, and mechanically oscillating to form microbubble ultrasound contrast agent containing leukocyte membrane, wherein the mass ratio of leukocyte membrane protein in leukocyte membrane to phospholipid in liposome is 1:50-600, and the gas is perfluoropropane C 3 F 8 。
2. The microbubble ultrasound contrast agent according to claim 1, wherein the phospholipid comprises one or more of Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA).
3. The microbubble ultrasound contrast agent according to claim 1, wherein the phospholipid comprises a combination of Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA).
4. The microbubble ultrasound contrast agent according to claim 1, wherein the phospholipid comprises Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA) in a molar mass ratio of 18: 1:1 to 5.
5. The microbubble ultrasound contrast agent according to claim 1, wherein the phospholipid comprises Dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylethanolamine-polyethylene glycol 2000(DSPE-PEG2000), and dipalmitoylphosphatidic acid (DPPA) in a molar mass ratio of 18: 1: 1.
6. the microbubble ultrasound contrast agent according to claim 1, wherein the mass ratio of leukocyte membrane protein to phospholipid is 1: 50.
7. The microbubble ultrasound contrast agent according to claim 1, wherein the microbubble ultrasound contrast agent has a size of 0.2-4.4 μm.
8. The microbubble ultrasound contrast agent according to claim 1, comprising the steps of:
s1, separating blood cells from peripheral blood;
s2, extracting cell membranes by using a differential centrifugation method;
s3, preparing liposome by a thin film-hydration method;
and S4, mixing the cell membrane prepared in the S2 with the liposome prepared in the S3, introducing gas, and mechanically oscillating to form the microbubble ultrasound contrast agent containing the cell membrane.
9. The microbubble ultrasound contrast agent according to claim 8, wherein step S1 includes the steps of:
s11, taking a blood sample in an anticoagulation blood collection tube;
s12, adding the blood cell separation liquid with the same volume as the blood sample in the S11 into a centrifuge tube, sucking the blood sample, paving the blood sample on the liquid surface of the cell separation liquid, and centrifuging for 20-40min at 400-1000g at 18-22 ℃;
s13, after centrifugation in the step S12, sucking white blood cells in the separation liquid;
s14, adding lysis solution with the volume 2-5 times that of the cells into the white blood cells sucked in the step S13, blowing, uniformly mixing, lysing for 5-20min, centrifuging for 5-15min at 200g-400g, and removing supernatant;
s15, repeating S14 to obtain the required leucocytes;
s16, adding 5-15ml of cell washing liquid into the white blood cells obtained in the step S15, uniformly mixing the cells, centrifuging for 5-15min at 200-400g, and removing the supernatant;
s17, repeating the step S16 for three times to obtain the purified white blood cells.
10. The microbubble ultrasound contrast agent according to claim 8, wherein step S2 includes the steps of:
s21, adding 10-20ml of hypotonic lysis solution into the white blood cells separated in the step S1, re-suspending, transferring into a glass homogenizer, and homogenizing for 15-30 times on ice to obtain homogenate;
s22, adding the homogenate obtained in the step S21 into a centrifuge tube, centrifuging for 15-30min at 10,000g, sucking supernatant, centrifuging for 30-50min at 100,000g, discarding the supernatant, and keeping precipitate;
s23, re-suspending the precipitate obtained in the step S22 into a solution by using triple distilled water, freezing the re-suspended solution at-70 to-90 ℃, and performing vacuum freeze-drying to obtain the leucocyte membrane, and storing at-15 to-25 ℃.
11. The microbubble ultrasound contrast agent according to claim 10, wherein in the step S21, the hypotonic lysis solution comprises 30mM ph7.5 Tris-HCl, 225mM D-mannitol, 75mM sucrose, 0.2mM EGTA, a protease inhibitor and a phospholipase inhibitor.
12. The microbubble ultrasound contrast agent according to claim 10, wherein in step S23, the DiI dye is added when the precipitate obtained in step S22 is resuspended in a solution with triple-distilled water.
13. The microbubble ultrasound contrast agent according to claim 8, wherein step S3 includes the steps of:
s31, mixing phospholipid and chloroform;
s32, forming a phospholipid film by the solution prepared in the S31 under the vacuum action of a rotary evaporator;
s33, adding a hydration liquid into the phospholipid film prepared in the S32, and hydrating the phospholipid film in a rotary evaporator under a hot water bath to form liposome; the hydration solution is selected from double distilled water, sodium chloride or Phosphate Buffer Solution (PBS).
14. The microbubble ultrasound contrast agent according to claim 13, wherein the ratio of hydration fluid to phospholipid is 3-8ml to 20 mg.
15. The microbubble ultrasound contrast agent according to claim 13, wherein in step S31, the ratio of chloroform to phospholipid is 3-8ml to 20 mg.
16. The microbubble ultrasound contrast agent according to claim 13, wherein the dye DiO is added to the liposome prepared in S33.
17. The microbubble ultrasound contrast agent as claimed in claim 13, wherein in step S32, the rotational speed of the rotary evaporator is 100-120rpm, and the temperature is 50-70 ℃.
18. The microbubble ultrasound contrast agent according to claim 13, wherein the temperature of the rotary evaporator is 60 ℃ in step S32.
19. The microbubble ultrasound contrast agent according to claim 13, wherein in step S33, the hydration temperature is 50-60 ℃ and the hydration time is 20-40 min.
20. The microbubble ultrasound contrast agent according to claim 13, wherein the hydration time is 30min in step S33.
21. The microbubble ultrasound contrast agent according to claim 8, wherein step S4 includes the steps of:
s41, gas substitution: mixing the liposome prepared in the step S3 with the cell membrane prepared in the step S2, and introducing gas to replace air;
and S42, mechanically shaking the mixed solution obtained in the step S41 for 40-50S to form the microbubble ultrasound contrast agent.
22. The microbubble ultrasound contrast agent according to claim 21, wherein in step S42, the mixed solution is mechanically shaken for 45S.
23. The microbubble ultrasound contrast agent according to claim 21, wherein in the step S42, the mechanical oscillation frequency is 3000-5000 cpm.
24. The microbubble ultrasound contrast agent according to claim 21, wherein in step S42, the mechanical oscillation frequency is 4350 cpm.
25. The microbubble ultrasound contrast agent according to claim 21, wherein in step S41, the ratio of gas introduced to liposomes is 5-15: 0.5 ml.
26. The microbubble ultrasound contrast agent according to claim 21, wherein in step S41, the ratio of gas introduced to liposomes is 10: 0.5 ml.
27. Use of a microbubble ultrasound contrast agent as claimed in any one of claims 1 to 26 in the preparation of a targeted ultrasound contrast agent.
28. The use of claim 27, wherein the microbubble ultrasound contrast agent is targeted to a site of inflammation.
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| CN113425863A (en) * | 2021-07-01 | 2021-09-24 | 南京邮电大学 | Preparation method of platelet membrane-liposome fusion membrane microbubbles and application of platelet membrane-liposome fusion membrane microbubbles in thrombus ultrasonic molecular imaging |
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